CA1110185A

CA1110185A - Production of bacterial cell aggregate

Info

Publication number: CA1110185A
Application number: CA320,470A
Authority: CA
Inventors: Gerald B. Borglum
Original assignee: Miles Laboratories Inc
Current assignee: Bayer Corp
Priority date: 1978-03-27
Filing date: 1979-01-30
Publication date: 1981-10-06
Also published as: IT1114710B; RO78777A; AU512660B2; DK122379A; NL188359B; YU73279A; DE2911557B2; DE2911557C3; IL56732A; NL7902083A; DK151637C; HU182535B; JPS5715879B2; GB2033396A; BE875032A; ATA223579A; FR2421213A1; NO790984L; IL56732A0; AR222482A1

Abstract

PRODUCTION OF BACTERIAL CELL AGGREGATE ABSTRACT OF THE DISCLOSURE A bacterial cell aggregate having increased particle hardness is produced by contacting a mass of such bacterial cells with a cross-linking reaction product of (1) glutar-aldehyde, cyanuric halide or combinations thereof and (2) a specific cationic polymer obtained by polymerization of an epihalohydrin and an alkylene polyamine, recovering the resulting aggregate and drying the aggregate. -1-

Description

BACKGROUND AND PRI OR ART

Glucose isomerase is an enzyme that can be employed to catalyze the conversion of glucose (dextrose) to -fructose (levulose). It is known tha.t glucose isomerase can be produced by fermentation of certain organisms, such as Streptomyces flavovirens 9 Streptomyces echinatur, -Stre~tomyces achromogenus, Streptomyces albus~ _r~
myces olivaceus 7 Bacillus coagulans and the like~ in appropriate nutrient media. The glucose isomerase is formed inside the bacterial cells which grow dur;.ng its production. The cel:ls can be Eiltered off Erom the fermentation beer and used directly as a source of glucose isomerase. Direct commercial use of such enzyme-containing bacterial cells had been hampered, however, by a major disadvantage. The enzyme activity was lost from the cells during use and thus the useful life o the cells was re-duced. This disadvantage was overcome by the treatment of the bacterial cells with glutaraldehyde as described in U.S.
Patent No. 3,779,869. Additional techniques for immo-bilizing the enzyme acti~ity in bacterial cells as well as for forming aggregates of such enzyme-containing bacterial cells are described -for example, in U.S. Patent No.
3,821,086 and its Reissue U.S. Patent Nos. 29,130 and 29,136 and in South A-frican Patent No. 73/5917. The above U.S. patents relate to use o-f certain anionic and cationic polyelectrolyte flocculating agents. The South African patent discloses various combinations of binders, rein-forcing agents and cross-linking agents. While the above :

- 2 1 techniques provided bacterial cell aggregates which generally retained their enzyme ac~ivity during use, there was still a need -to increase the hardness of the aggregates so that they could be commercially used in reactor beds of increasing depth. U.S. Patent No. 3,935,069 describes the addition of cer-tain metallic compounds in conjunction with - polyelectrolyte flocculating agents to improve the hardness.
However, this technique has limited utility.

SUMMARY OF THE INVENTION

In accordance with the present invention, a process is provided or the production of an aggregate of bacterial cells having improved hardness. This process i.nvolves the use of a cross-linking reaction product of glutaraldehyde and/or cyanuric halide and a particular epihalohydrin -polyamine polymer. In particular, this invention relates to a process for producing an aggregate o-f bacterial cells which comprises contacting a mass of bacterial cells with a cross-linking reaction product of ~1) a material selected from the class consisting of glutaraldehyde, cyanuric halide and combinations thereof and ~2) a water-solu~le cationic polymer obtained by the polymerization of an epihalohydrin with an alkylene polyamine having the formula RlR2NRNH2 wherein R is a lower alkylene having from Z to about 6 carbon atoms, and Rl and R2 are each a lower alkyl of from 1 to about 6 carbon atoms, the mole ratio of epihalohydrin to polyamine being from about 0.60:1 to about 2.7:1, said polymerization comprising 1 reacting with the alkylene polyamine from about 50 to about 90 percent of the amount of epihalohydrin ~o be polymerized, allowing the reaction to continue until the reaction medium attains a substantially uniform viscosity, and reacting the remaining portion of the epihalohydrin incrementally to obtain the cationic polymer, the temper-ature of polymeri7ation being -from about 60C. to about 120C., and recovering the resulting aggregate. This invention is especially useful when the resulting aggre-gate is dried and then rehydrated for subsequent use.

DFSCRIPTION OF THE INVENTION

The process o the pr0sent invention can be used with various enzyme-containing bacterial cells. The remainder of the disclosure will be directed at using the process with bacterial cells containing glucose isomerase activity.
The bacterial cells containing glucose isomerase activity useful in the process of the present invention can be produced by well-known procedures. The preferred enzyme-containing cells are produced by growing under submerged aerobic conditions a culture of Streptomyces olivaceus NRRL 3583 or mutants thereof in a medium con-taining appropriate nutrien~s. This is described in U.S.
patent No. 3,625,828. The resulting bacterial cells are separated -from the fermentation beer by -filtration or ; 25 centrifugation.
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1 The i.ngredients employed in this process are readily available. Glutaraldehyde and cyanuric halide, such as cyanuric trichloride, cyanuric tri.bromide, cyanuric tri-iodide and the like, are commercially available or can be produced by well-known techniques. The particular ; epihalohydrin-polyamine polymer used in this process is : commercially available under the trademarX ~ETZ 1180 from Betz Laboratories, Inc. Trevose, Pennsylvania. BP.TZ 1180 has a molecular weight less than one million, contains about 0.288 millimoles of amino groups per gram of solu-tion ~based on a ninhydrin assay) and is marketed as a solution containing 30 weight percent solids 9 based on -total solution weight. This compound is disclosed in U.S~ Patent No. 3,915,904. The compound is described therein as a water-soluble cationic polymer obtaine(l by the polymerization of an epihalohydrin with an alkylene polyamine having the formula RlR2MRNH2 wherein R is a .-.-.
lower alkylene having from 2 to about 6 carbon atoms, and Rl and R2 are each a lower alkyl of from about 1 to about 6 carbon atoms, the mole ratio of epihalohydrin to poly-amine being :trom about 0.60:1 to about 2.7:1, said polymerization comprising reacting with the alkylene polyamine from about 50 to about 90 percent of the amount of epihalohydrin to be polymerized, allowing the reaction to continue until the reaction medium attains a substan-tially uniform viscosity9 and reacting the remaining pOT-tion of the epihalohydrin incrementally to obtain the cationic polymer~ the temperature of polymerization being from about 60C. to about 120C. This material will here-ina~ter be referred to as the "polyamine polymer".

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1 The cross-linking reac~ion product employed in the present invention to form the bacterial cell aggregate can be one of three possible compositions. The polyamine polymer can be reacted with glutaraldehyde or cyanuric halide or with both glutaraldehyde and cyanuric halide.
The glutaraldehyde and/or cyanuric halide, which is collectively identified as component (1), is reacted with the polyamine polymer, which is identified as component ~2), at a pH about 6 to 10 and at about 0 to 30C. for about 0.5 to 2.5 hours. The overall cross-linking reaction product contains from about 12 to about 77 weight percent of component ~1) and from about 23 to about B8 weight percent of component (Z) based on the total weight of the active ingredients in components ~1) and (2). The glutaraldehyde content of the reaction product is from about 0 to about 77 weight percent and the cyanuric halide content is from about 0 to about 22 weight percent based on the total weight of the active lngredients in com-ponents (1) and (2).
The reaction between glutaraldehyde and the polyamine polymer is preferably carried out at pH 8 to 9 and at about 18 to 25C. :Eor about 0.5 hour. The glutaraldehyde should be present in a molar ratio of at least one mole ~ per mole of amino group in the polyamine polymer in order ; 2S to avoid undesirable cross-linking of the polyamine polymer with glutaraldehyde.
The reaction between cyanuric halide alone and the polyamine polymer is preferably carried out at pH 8 to 9 and at 0 to 10C. for about 1 to 2 hours. The cyanuric - G -1 halide should be present in a molar ratio o-f at leas~
one mole per mole of amino group in the polyarnine polymer in order to avoid undesirable cross-linking o-f the poly-amine polymer with cyanuric halide. Cyanuric halide, such as cyanuric trichloride, has three halogen reactive sites.
~ne of these sites will react at 0C. or higher. After reaction at the irst site, the second site will react at 30 to 50C. and the final site will react at 90 to 100C. It is desirable to initia~ly react only the -first site on the cyanuric halide ~ith the polyamine polymer.
When the resulting cross linking reaction product is subsequently reacted with the bacterial ce:Lls and heated to higher telllperatures during drying, the remaining re-; active sites on the cyanuric halide will then react with the polyamine polymer to provide additional cross-linking to the bacterial cell aggregate.
The reaction between the polyamine polymer and the combination of glutaraldehyde and cyanuric halide is carried out in steps. First, the cyanuric halide is re-acted with the polyamine polymer at pH 8 to 9 and at 0 to 10~. for about 1 to 2 hours. Preferably, in this situation the reactants have a mole ratio oE one mole of cyanuric halide to two moles o amino groups on the polyamine polymer. An excess amount of glutaraldehyde is then added and the reaction is continued under the same p~ and temperature conditions for about 0.5 hour.
The cross-linking reaction product employed in the present invention is not a cationic polyelectrolyte, since the amino groups on the polyamine polymer which initially l provided the cationic characteristic have been reacted with the glutaraldehyde and/or cyanuric halide and are thus no longer available.
Bacterial cell aggregates are prepared by contacting a mass o-f bacterial cells with the cross-linking reaction product prepared as described above at pH about 8 to 9 and at about 0 to 30C. -for about 0.5 to 1.5 hours. The cross-linking reaction product is employed in such amount ~ and concentration that the bacterial cells are contacted ; 10 with from about 4.5 to about 60 weight percent of the cross -linking reaction product active ingredients based UpOII
the dry weighk oE the cells.
After the above reaction takes place, the resulting bacterial cell aggregate is preferably extruded or other-wise formed into desirable shapes and the dried at about 65C. for several hours. The resulting dried aggregate can be stored until subsequently needed -~or use in an enzymatic process. At that time the dried aggregate is rehydrated and conditioned for use. One illustrative conditioning process is described in U.S. Patent No.

3,974,036.
A principal advantage o~ the present invention is an increase in the hardness o~ ~he bacterial cell aggregate after rehydration as compared -to prior art bacterial cell aggregates. The hardness is expressed in relation to resistance to compression o~ the bacterial cell aggre-gate particles. An Instron Tensile Tester using a Com-pression Load Cell No. CCT was employed in a manner similar to that described in U.S. Patent No. 3,935,069.
This instrument is available from Instron Corporation, Canton, Massachusetts.
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LO~L85 1 The following is the Rehydration ~lardness Assay Procedure:
A rehydration solution is prepared by mixing 9.68 g CoC12 6H2O, 28.0 g Mg~OH)2 and 56.0 g anhydrous citric acid in 600 ml. distilled water at 45C. The mix~ure is stirred and heated to 60C. to dissolve all -the salts. It is then cooled to 25C. and adjusted to pH 8.5 with NaOH.
It is then filtered and brought to 1.0 1. volume with distilled water. A 2.5 ml. portion of the above solution is mixed with 130 ml. water, 70.3 g dextrose, 24.228 g tris (hydroxymethyl) aminomethane and adjusted to pH 8.55 at 25C. with NaOH. It is then brought to 200 ml. with distilled water.
~ive particles o-f dried bacterial cell aggrega-te are covered with 2 5 ml. of the above rehydration solution in a petri dish and heated at 60C. in a water bath for one hour and then allowed to stand at room temperature until cooled. The particles are removed from the solution, excess surface liquid is removed, and then they are tested on the Instron instrument. The instrument is warmed up Eor at least 30 minutes with the load cell attached before being used for measurements. Set crosshead speed at 0.2 in/min. ~5.1 mm./min.) and the chart speed at 2.0 in./min. (51 mm./min.). Set the "Return" to halt at 0.038 in. ~0.965 mm.) for the surface of the load cell.
Set "Gage Length" to clear the lip of the sample cup.
Set the recorder to full scale range. This is usually 10 pounds ~4.54 kg.). Standardize the recorder to read zero pounds ~or zero kg.) with -the sample cup on the load cell :.

g 1 and one pound ~0.454 kg.) with the cup and one pound ~0.454 ~g.) standard weight on the load cell. Place a single rehydrated particle on the sample cup centered with the crosshead. Manually lower the crosshead to the top of the particle and press the "Run" button. On the recorder read the force in pounds (~g.) at a distance of 0.03 in. ~0.762 mm.) from the point at which the cross-head touches the particle. The hardness is thus expressed in the ~orce (pounds or kilograms) needed to compress the particle 0.03 in. (0.762 mm.). The test is repeated for several particles and the results are averaged.
The invention is described in further detail in the following illustrative examples.

EX~MPL~ 1 A cross-linking reaction product was obtained by ; adding 2.25 g. BETZ 1180 solution containing 0.675 g.
active material and 0.648 millimoles amino groups to 100 ml. of 1.25 percent (weight/volume basis) glutaralde-'~ hyde containing 13.24 millimoles active material at p~l 9.
The mixture was stirred for about 30 minutes and became a deep yellow solution. The resulting product was formed from a reaction mixture containing 64.9 weigh~ percent glutaraldehyde and 35.1 weight percent polyamine polymer based on total weight of the glutaraldehyde (Component 1) and the polyamine polymer ~Component 2).

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1 A culture of a mutant of Streptomyces olivaceus NRRL 3583 was grown in an agitated aerated fermentor con-taining an appropriate nutrient medium described in U.S.
Patent No. 3,625,828. The resulting fermentor broth con-taining a mass of bacterial cells was adjusted to pH 8~2 by addition of appropriate buffering materials. A portion of the above-prepared solution was added to a portion of the fermentor broth in an amount to provide the equivalent of 14 weight percent glutaraldehyde (21.6 weight percen~ tota]
re~ction product) based on the dry weight of the bacterial cells. After 30 minutes reaction time at 25C. and p~l 8.2, the treated broth was filtered. The filter cake was then extruded through a syringe opening of 2.2 mm. The result-ing extruded strands were cut into indiviclual 30 mm. lengths and dried overnight at 65C. in a ~orced warm air oven. A
similar portion of ~ermentor broth was treated with glu-taraldehyde alone at a concentra-tion of 14 weigh~ percent based on bacterial cell dry weight. The treated cells were then -filtered, extruded and dried in the same manner to produce Control particles. Both the Control and the cross -linking reaction product treated materials were tested for hardness. The Control sample had a hardness of 0.8 lb.
(0.364 kg.) while the product prepared in accordance with the present invention had the improved hardness o~ 2.8 lb.
; 25 ~1.27 kg.).

Portions o S _ ptomyces olivaceus fermentor broth similar to that of Example 1 were treated with glutaralde-hyde alone (Control) and with various combinatiorls of 1 cross-linking reaction products at pH 9 and 29C. The various cross-linking reaction products were prepared as described in Example 1 above using various amounts of glutaraldehyde and polyamine polymer. The treated bacterial cells were then filtered~ extruded, dried and tested for hardness, The results are shown in the ; following Table I.

TABLE

Reaction Mixture for Composition of Cross -Linking Reaction Product (weight percent) Amount Added Glutar- Polyamine ~Weight }lardness aldehyde Polymer Percent) lb~(kg.) _ 100 (Control) 13.9 1.8 (0.82) 76.7 23.3 13 2.7 (1.23) ~O.o 60.0 34.7 2.7 (1.23) 44.4 55.6 18.7 3.3 (1.5) 65.~ 34.9 29.8 3.6 (1.6~) It can be seen that the use of the cross-linking reaction product enables consistently increased hardness to be obtained as compared to the prior art use of glutaraldehyde alone.

A cross-linking reaction product was obtained by dissolving 0.188 g cyanuric trichloride (0.64 millimoles) in l0 ml. acetone and then adding this solution with stirring to 70 ml. ice-cold water to give a finely -divided precipitate. A 2.25 g portion oE BETZ 1180 1 solution (containing 0.675 g active material and 0.648 millimoles amino groups) was diluted with 20 ml. water and added to the cyanuric trichloride suspension. The resulting mix~ure was stirred and maintained at pll 9 and 0-5C. for 1-2 hours, then diluted to 100 ml. The cyanuric trichloride dissol~ed indicating reac-tion with the polyamine. This reaction product resulted frorn a reaction mixture containing 21.8 weight percen~ cyanuric trichloride as component (1) and 78.2 weight percent polyamine polymer as component (2). A portion of a Streptomyces olivaceus Eermentor broth similar to that oE ~ample 1 was mixed with a portion o~ the abovc re-action product to provide a concentration of 32.0 weigllt percent reaction product based on dry weight o the bacterial cells. A 0.2 percent ~weight/volume basis) aqueous sodium bicarbonate solution was added to main-tain pH at 9. After 1.5 hours at pH 9 and 25CC. the treated broth was iltered, extruded and dried as de-scri'bed in Example 1. Another portion of fermentor broth was treated as above :Eor 0.5 hour. No sodium bicarbonate was initially added, but the Eiltered cells were washed with 1 weight percent sodium 'bicarbonate solution at pH 9 before extruding and drying. For a Control, glu-taraldehyde was added to a separate portion of the Z5 -fermentor broth at a concentration o 14 weight percen-t based on the dry weight o the bacterial cells. The cells were treated with glutaraldehyde for 30 minutes - at pH 8.2 and 25~C. beore iltering, extruding and drying. The Control produced a hardness oE 2.2 lb. (1 kg.) 1 while -~he 0.5 hour reaction product treatment produced a hardness of 3.8 lb (1.73 kg.) and the 1.5 hours reaction product treatment produced a hardness of 4O0 lb (1.82 kg.).

A cross-linking reaction product was obtained by adding 4.5 g BETZ 1180 solution (containing 1.35 g active material and 1.296 millimoles of amino groups) to 0.118 g (0.64 millimoles) of finely-divided cyanuric trichloride în ice-cold water. The pH was adjusted to 9 and was maintained at pH 9 in an ice-ba-th (0C.) for two hours. Then l.25 g. (13.24 millimoles) o~ glutaral-dehyde at pH 9 was added and the low temperature main-tained for about 0.5 hour. A dark yellow color de~eloped.
The reaction product resulted from a reaction mixture con-taining 4.3 weight percent cyanuric trichloride, 46.0 weight percent glutaraldehyde (total of 50.3 weight percent component 1) and 49.7 weight percent polyamine polymer as component (2). A portion of a Streptomyces olivaceus fermentor broth similar to that of Example 1 was mixed with a portion of the abo~e reaction product to provide a concentration of 30.4 weight percent reaction product based on dry weight of the bacterial cells. A~ter a 0.5 hour reaction period at pH 9 and 25C. the treated broth was filtered, washed with 5 weight percent aqueous sodium bicarbonate solution at pH 9, extruded and dried.
A Control sample was prepared in -the manner described in Example 3. The Control produced a hardness of 0.8 lb.
~0.364 kg.) while the cross-linking reaction product procluced a hardness o-f 3.8 lb. ~1.73 kg.).
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Portions of Streptomyces olivaceus fermentor broth similar to that of Example 1 were -treated with glutaral-dehyde alone ~Control) and with various combinations of reaction products at pH 9 and at 25C. and 5C. The cross-linking reaction products all employed one mole of cyanuric trichloride per two moles of amino groups in the polyamine polymer. The overall amounts of glutaraldehyde and polyamine polymer were then adjusted .to produce the combinations set forth in Table II below.
The treated bacterial cel:Ls were then filtered, extruded, dried and tested ~or harclness. The results are shown i the following Table II.

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C~l ~1 un o un s 1 It should be noted that in all the above examples the bacterial cells treated with the cross-linking re-action products all had significantly increased hardness ~alues as compared to the bacterial cells treated with the prior art gluta~aldehyde alone.
When a cross-linking reaction product produced from glutaraldehyde and polyamine polymer is used, the pre-ferred composition is produced from a mixture of 57.1 weight percent glutaraldehyde as c~mponent ~1) and 42.9 weight percent polyamine polymer as component (Z) based on the total weight of the active ingredients in components (1) and (2). This composition is also pre~era~ly employed in an amount o~ 17.5 weight percent based on the dry weight o~ the bacterial cells.
When a cross-linking reaction product produced from glutaraldehyde, cyanuric trichloride and polyamine polymer is used, the preferred composition is produced from a mix-ture o~ 54.9 weight percent glutaraldehyde and 3.6 weight percent cyanuric trichloride as component (1) and 41.5 weight percent of polyamine polymer as component (Z) based on the total weight of the active ingredients in components (1) and (2). This composition is also preferably employed in an amount of 18.2 weight percent based on the dry weight of the bacterial cells.
, 25 The bacterial aggregates produced in the manner de-cribed abo~e were all capable of converting glucose to fructose. The glucose isomerase activity was not impaired through the use of this novel process.

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Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for producing an aggregate of bacterial cells which comprises contacting a mass of bacterial cells with a cross-linking reaction product of (1) a material selected from the class consisting of glutaraldehyde, cyanuric halide and combinations thereof and (2) a water -soluble cationic polymer obtained by the polymerization of an epihalohydrin with an alkylene polyamine having the formula R1R2NRNH2 wherein R is a lower alkylene having from 2 to about 6 carbon atoms, and R1 and R2 are each a lower alkyl of from l to about 6 carbon atoms, the mole ratio of epihalohydrin to polyamine being from about 0.60:1 to about 2.7:1, said polymerization comprising reacting with the alkylene polyamine from about 50 to about 90 percent of the amount of epihalohydrin to be polymerized, allowing the reaction to continue until the reaction medium attains a substantially uniform viscosity, and reacting the remaining portion of the epihalohydrin incrementally to obtain the cationic poly-mer, the temperature of polymerization being from about 60°C. to about 120°C., and recovering the resulting aggregate.

2. A process according to Claim 1 wherein component (1) of the cross-linking reaction product is glutaral-dehyde.

3. A process according to Claim l wherein com-ponent (1) of the cross-linking reaction product is cyanuric halide.

4. A process according to Claim 1 wherein com-ponent (1) of the cross-linking reaction product is a combination of glutaraldehyde and cyanuric halide.

5. A process according to Claim 1 wherein the bacterial cells are contacted with the cross-linking reaction product at pH about 8 to 9 and at about 0° to 30°C. for about 0.5 to 1.5 hours.

6. A process according to Claim 1 wherein the bacterial cells are contacted with from about 4.5 to about 60 weight percent of the cross-linking reaction product based upon the dry weight of the cells.

7. A process according to Claim 1 wherein the cross-linking product results from the reaction of from about 12 to about 77 weight percent of component (1) and from about 23 to about 88 weight percent of com-ponent (2) based on the total weight of the active ingredients in components (1) and (2).

8. A process according to Claim 7 wherein component (1) of the cross-linking reaction product con-tains from about 0 to about 77 weight percent glutaralde-hyde and from about 0 to about 22 weight percent cyanuric halide and wherein the total amount of glutaraldehyde and/or cyanuric halide is from about 12 to about 77 weight percent, said weight percents based on the total weight of the active ingredients in components (1) and (2).

9. A process according to Claim 1 wherein the bacterial cells are Streptomyces olivaceus.

10. A process according to Claim 1 wherein the cyanuric halide is cyanuric trichloride.

11. A process according to Claim 1 wherein the cross -linking product results from the reaction of 57.1 weight percent glutaraldehyde as component (1) and 42.9 weight per-cent of component (2), based on the total weight of the active ingredients in components (1) and (2), and such cross--linking reaction product is used in an amount of 17.5 weight percent based on the dry weight of the bacterial cells.

12. A process according to Claim 1 wherein the cross--linking product results from the reaction of 54.9 weight percent glutaraldehyde and 3.6 weight percent cyanuric tri-chloride as component (1) and 41.5 weight percent of com-ponent (2), based on the total weight of the active ingre-dients in components (1) and (2), and such cross-linking reaction product is used in an amount of 18.2 weight per-cent based on the dry weight of the bacterial cells.

13. A process according to Claim 1 wherein the cross-linking reaction product is obtained by reacting components (1) and (2) at a pH about 6 to 10 and about 0° to 30°C. for about 0.5 to 2.5 hours.

14. A process according to Claim 1 wherein the recovered aggregate is then dried.

15. A process for producing an aggregate of Streptomyces olivaceus bacterial cells which comprises contacting a mass of such bacterial cells at pH about 8 to 9 and at about 0° to 30°C. for about 0.5 to 1.5 hours with from about 4.5 to about 60 weight percent, based on the dry weight of such cells, of a cross-linking product resulting from the reaction of (1) from about 12 to about 77 weight percent of a material selected from the class consisting of glutaraldehyde, cyanuric trichloride and combinations there-of and (2) from about 23 to about 88 weight percent of a water-soluble cationic polymer obtained by the polymeriza-tion of an epihalohydrin with an alkylene polyamine having the formula R1R2NRH2 wherein R is a lower alkylene having from 2 to about 6 carbon atoms, and R1 and R2 are each a lower alkyl of from 1 to about 6 carbon atoms, the mole ratio of epihalohydrin to polyamine being from about 0.60:1 to about 2.7:1, said polymerization comprising reacting with the alkylene polyamine from about 50 to about 90 percent of the amount of epihalohydrin to be polymerized, allowing the reaction to continue until the reaction medium attains a sub-stantially uniform viscosity, and reacting the remaining portion of the epihalohydrin incrementally to obtain the cationic polymer, the temperature of polymerization being from about 60°C. to about 120°C., said weight percents of components (1) and (2) being based on the total weight of the active ingredients in components (1) and (2), said reaction between components (1) and (2) taking place at a pH about 6 to 10 and about 0° to 30°C. for about 0.5 to 2.5 hours, and recovering the resulting aggregate.

16. A process according to Claim 15 wherein component (1) of the cross-linking reaction product is glutaraldehyde.

17. A process according to Claim 15 wherein component (1) of the cross-linking reaction product is a combination of glutaraldehyde and cyanuric trichloride.

18. A process according to Claim 15 wherein the cross -linking product results from the reaction of 57.1 weight percent glutaraldehyde as component (1) and 42.9 weight per-cent of component (2), based on the total weight of the active ingredients in components (1) and (2), and such cross -linking reaction product is used in an amount of 17.5 weight percent based on the dry weight of the bacterial cells.

19. A process according to Claim 15 wherein the cross -linking product results from the reaction of 54.9 weight percent glutaraldehyde and 3.6 weight percent cyanuric tri-chloride as component (1) and 41.5 weight percent of com-ponent (2), based on the total weight of the active ingre-dients in components (1) and (2), and such cross-linking reaction product is used in an amount of 18.2 weight percent based on the dry weight of the bacterial cells.

20. A process according to Claim 15 wherein the recovered aggregate is then dried.

21. An aggregate of bacterial cells produced by the process of Claim 1.